Abstract Current state-of-the-art treatment of rheumatoid arthritis (RA) is not curative and is associated with considerable toxicity. Substantial evidence now exists that thymus-derived, naturally occurring CD4+CD25+Foxp3+ regulatory T cells (nTreg) play an important role in the prevention of autoimmune diseases including RA. Reduced numbers and the dysfunctional nature of nTreg have been associated with development and progression of RA and other autoimmune diseases. Although adoptive transfer of nTreg to mice with autoimmune arthritis can significantly prevent the disease progression, the effect of these cells on established disease is less effective. The major reason is that nTregs are unstable and retain a plasticity that allows them to convert to Th17/Th1 effector cells, diminishing their functional activity. In the cycle of our previous grant, we have made two milestone observations: 1) unlike nTreg, iTreg induced ex vivo with IL-2 and TGF-? are stable and functional in conditions with inflammation; 2) nTreg primed with all-trans retinoic acid (atRA) are able to restore their stability and functionality for therapeutic use in the presence of inflammation and in established autoimmune diseases. Given that the inflamed synovial tissue and synovial fibroblast are main sources of pro-inflammatory cytokines and key players for joint damage in autoimmune arthritis, we will test in this renewal proposal whether iTreg maintain their phenotype and function in the inflamed synovial tissues and even suppress the proliferation, migration and cytokine production of inflamed synovial cells and tissues. We propose a set of experiments using conditional KO mice and humanized animal models that will allow us to understand the mechanisms whereby iTreg induced and nTreg modified display their stability and functionality in the inflammatory condition. We predict that DBC1, a molecule that we have identified to have a key role in this process, is crucial for Treg stability in the presence of inflammation. We will also study whether the findings regarding the approach for stabilizing mouse nTreg can be extended to human nTreg from patients with RA. To achieve these aims, we will develop the strategies with the following four specific aims to conduct specific experiments. Aim 1: examine the fates of iTreg in the presence of inflamed synovial tissue. Aim 2: determine whether crosstalk between iTreg and DC contributes to long-term effects of iTreg on CIA. Aim 3: determine whether atRA maintains phenotypic and functional stability of nTreg from RA patients and Aim 4: elucidate the in vitro and in vivo functional role of DBC1 in Treg of rheumatoid arthritis. When successfully completed, this project will help us understand the characteristics of Treg subsets in autoimmune arthritis and may lead to a potential therapeutic strategy for RA and other human autoimmune diseases.